Proteasome inhibitors prevent caspase-1-mediated disease in rodents challenged with anthrax lethal toxin

Abstract

NOD-like receptors (NLRs) and caspase-1 are critical components of innate immunity, yet their over-activation has been linked to a long list of microbial and inflammatory diseases, including anthrax. The Bacillus anthracis lethal toxin (LT) has been shown to activate the NLR Nalp1b and caspase-1 and to induce many symptoms of the anthrax disease in susceptible murine strains. In this study we tested whether it is possible to prevent LT-mediated disease by pharmacological inhibition of caspase-1. We found that caspase-1 and proteasome inhibitors blocked LT-mediated caspase-1 activation and cytolysis of LT-sensitive (Fischer and Brown-Norway) rat macrophages. The proteasome inhibitor NPI-0052 also prevented disease progression and death in susceptible Fischer rats and increased survival in BALB/c mice after LT challenge. In addition, NPI-0052 blocked rapid disease progression and death in susceptible Fischer rats and BALB/c mice challenged with LT. In contrast, Lewis rats, which harbor LT-resistant macrophages, showed no signs of caspase-1 activation after LT injection and did not exhibit rapid disease progression. Taken together, our findings indicate that caspase-1 activation is critical for rapid disease progression in rodents challenged with LT. Our studies indicate that pharmacological inhibition of NLR signaling and caspase-1 can be used to treat inflammatory diseases.

Figure 1

Caspase-1 activation is essential for LT-mediated necrosis in susceptible rat macrophages. A: F344 and LEW BMMs were subjected to LT (LF and PA: 250 and 500 ng/ml), and cell viability was determined by WST-1 assay. B and C: F344 BMMs were treated with LT in the presence or absence of 80 μmol/L Boc-D-CMK or 10 μmol/L MG132. Membrane integrity was measured by analysis of propidium uptake (B), and caspase-1 activation (C) was determined by FLICA assay as indicated in arbitrary units. D: LT triggers processing of IL-18 in F344 BMMs after LT exposure. Cleavage of pro-IL-18 was determined by Western blotting of lysates from these cells, and actin is shown as a loading control. Representative data are shown from at least three independent experiments. Data are represented as mean ± SD of N = 3. E: LT-treated F344 BMMs show signs of necrotic cell death, as analyzed by electron microscopy 4 hours post-LT exposure (Scale bars = 1 μm).

Figure 2

Strain-specific killing of rat macrophages. A: F344 and LEW BMMs are equally susceptible to LT killing. F344 and LEW BMMs were subjected to PA (500 ng/ml) and varying concentrations of LF. Cell viability of F344 and LEW BMMs was determined by WST-1 assay 2 and 48 hours post LT exposure, respectively. B: LT triggers caspase-3 in LEW macrophages, as determined by a colorimetric caspase-3 cleavage assay. C: F344 and LEW BMMs were exposed to LT and subjected to DAPI and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining 6 and 72 hours post-LT exposure, respectively. Representative data are shown from at least three independent experiments. Data are represented as mean ± SD of N = 3.

Figure 3

Proteasome inhibitors block caspase-1-mediated LT killing of F344 macrophages. A: The proteasome inhibitors MG132, NPI-0052, and bortezomib were applied at the given concentrations to F344 BMMs treated with LT. Viability was established by WST-1 assay 4 hours after LT-treatment. Data are represented as mean ± SD of N = 3, and representative data are shown from three independent experiments. B: The proteasome inhibitor NPI-0052 does not block caspase-1 directly. Lysates from LPS-primed J344 BMMs were incubated with recombinant caspase-1 in the presence of NPI-0052 and Boc-d-cmk for 2 hours. Western blots were performed with an IL-1β antibody to monitor caspase-1 activity. N.s. indicates nonspecific bands. Arrow points to mature IL-1β. C: NPI-0052 did not prevent caspase-1 activation and cytolysis of F344 BMMs mediated by the Nalp3-inducers LPS and nigericin. F344 BMMs were incubated with LPS and nigericin in the presence of NPI-0052 (uM) and Boc-d-cmk (uM) for 4 hours. IL-1β processing was measured by Western blotting and cell survival was determined by LDH release.

Figure 4

Proteasome inhibitors block LT killing in susceptible rat and murine strains. A: F344 rats (250 to 300 g) were injected IV with 1.0 mg/kg bortezomib (bortezomib only: N = 6) or 0.2 mg/kg NPI-0052 plus vehicle (NPI-0052 only: N = 6), followed by IV injection of either PBS or 0.15 mg/kg LT. Rat survival up to 240 minutes was measured following PBS (control: N = 5) or LT (LT only: N = 10, mean survival times: 59.7 minutes; LT + bortezomib, mean survival times: 135.4 minutes: N = 6; LT + 0052: N = 9) injections. Representative data are shown from three independent experiments. *P <0.05 for comparison with LT treatment alone. B: BALB/c mice were injected IV with NPI-0052 plus vehicle (200 μg/kg NPI-0052 only: N = 6; 250 μg/kg NPI-0052 only: N = 6), followed by IV injection of either PBS or 0.15 mg/kg LT (LT + 200 μg/kg NPI-0052: N = 10; LT + 250 μg/kg NPI-0052 only: N = 10). Survival up to six days was measured. Representative data are shown from three independent experiments. *P <0.05 for comparison with LT treatment alone.

Figure 5

NPI-0052 treatment prevents vascular defects induced by LT in F344 rats. A–C: F344 rats (250 to 300 g) were either mock injected with vehicle and PBS (control: N = 5), vehicle and LT (N = 9), NPI-0052 (N = 6) and PBS, or NPI-0052 and LT (N = 10). Moribund animals, present only in the LT only group, were sacrificed on loss of righting reflex; surviving animals were sacrificed four hours post injection. Blood was collected, and hematocrit measured (A). Clear pleural effusions were drained and quantified from opened thoracic cavities (B). Left lungs were removed, blotted on tissue paper, and weighed (C). D: Left lungs of untreated (PBS), LT-treated and LT/NPI-0052-treated rats were then preserved and analyzed for pulmonary edema by microscopy. Representative data are shown from at least three independent experiments. Scale bars = 100 μm. *P <0.05 for comparison with LT treatment alone.

Figure 6

Macrophage depletion in LT-treated F344 rats. A–E: F344 rats (250 to 300 g) were either injected with vehicle and PBS, vehicle and LT, NPI-0052 and PBS, or NPI-0052 and LT. Blood serum (A and B) and spleens (C–E) were collected 80 minutes post-LT injection. A and B: Levels of tumor necrosis factor and IL-18 were determined by Luminex-based multiplex assay. Representative data are shown from at least two independent experiments and are represented as ± SD of N = 3. *P <0.05 for comparison with LT treatment alone. C: Representative flow cytometry plots of untreated and LT-treated F344 rats are shown and gates are drawn around live splenic macrophages (CD3⁻, ED1^int, CD11b/c⁺). D and E: The percentage of macrophages (ED1⁺, CD11B/C⁺) and T cells (CD3⁺) were measured relative to controls. Macrophage and T cell populations were determined in untreated and LT-treated F344 rats (D), as well as in NPI-0052 and LT/NPI-0052-treated F344 rats (E). Representative data are shown from two independent experiments and are represented as ± SD of N = 3. *P = 0.001 for comparison with LT treatment.